Plasmid-directed gene expression is now near the efficacy barrier that to date has prevented commercialization of plasmid-based therapies for human health applications. Previous innovations such as electroporation (EP) increased transgene expression through more effective gene delivery. In Phase I we developed potent minimalized antibiotic-free expression vectors, and demonstrated dramatic improvements in transgene expression may be obtained through vector design innovations. Two platform technologies were developed. Novel compositions that prevent vector-backbone mediated transgene silencing from plasmid vectors (Anti-Silencing Elements: ASE platform) Novel vector backbone functionalities that improve transgene expression from plasmid vectors after transient transfection (transient expression enhancers: TEE platform) In Phase II we hypothesize that combining ASE and TEE vectors with state of the art plasmid delivery will create enabling vector-delivery platforms for gene therapy. In Specific Aims 1 and 2 optimal ASE/TEE antibiotic-free vector - EP delivery platforms for skeletal muscle and cutaneous gene therapy, respectively, are identified. In Specific Aim 3 the cutaneous gene therapy platform is applied to create a hypoxia-inducible factor 11 (HIF-11) based gene medicine for diabetic foot ulcer treatment. In Specific Aim 4 a dermatological gene therapy to treat skin aging is developed using a keratinocyte growth factor (KGF) vector- microdermabrasion delivery combination. Specific Aims 3 and 4 are performed in collaboration with wound healing gene therapy expert Dr. John Harmon at Johns Hopkins University and dermatology gene therapy expert Dr Aaron Tabor at Gene Facelift, LLC. The vector-delivery platforms developed herein will further improve gene expression to levels that will enable gene medicine licensure for multiple applications for unmet public health needs. In Phase III the gene therapies for diabetic foot ulcers and skin cosmetics will undergo clinical development. PUBLIC HEALTH RELEVANCE: The objective of this proposal is to validate a novel antibiotic-free non-viral gene therapy platform, and as such is responsive to NIGMS SBIR high-priority area of interest in development of improved vectors for gene transfer. The vectors contain transient expression enhancers that improve transgene expression level and duration after gene delivery to skin or muscle. The platform will be applied to create gene therapy products to treat diabetic neuropathic foot ulcers and skin aging.